Relative HPV Viral Load Measured by ΔCt for Risk Stratification in Cervical Cancer Screening

Background/Objectives: Human papillomavirus (HPV) testing is widely used in cervical cancer screening because of its high sensitivity, although its limited specificity requires effective triage strategies. This study aimed to evaluate the association between relative viral load (RVL) and cervical lesion severity, focusing on HPV-positive women with non-high-grade cytology. Methods: The study included 997 women with histological outcomes from a cohort of 2765 HPV-positive individuals identified among 37,030 women screened in the Bari metropolitan area (Italy) using a real-time HPV-DNA test. Associations between RVL, estimated using the ΔCt method, and lesion severity were assessed using histological and cytological classifications. Logistic regression models evaluated the independent effect of RVL, adjusting for age and HPV detection channel. Results: Lower ΔCt values (indicating higher RVL) were significantly associated with high-grade lesions (CIN2+) (OR = 0.91, 95% CI: 0.89–0.94, p < 0.001). The association was observed for HPV16 and the pooled 12-high-risk HPV detection channel, while no significant association was observed for HPV18. A similar association was observed when cytological classification was used as the outcome. Age showed a modest effect, with reduced odds of high-grade lesions observed in women aged ≥50 years. Among women with non-high-grade cytology who subsequently underwent histological assessment, 17.6% had underlying CIN2+, and RVL remained significantly associated with lesion severity in this subgroup. However, ΔCt showed only modest discrimination for high-grade lesions [AUC = 0.643 (95% CI: 0.605–0.681)]. Conclusions: Higher HPV RVL is associated with increased lesion severity, particularly for HPV16 and the pooled 12-high-risk detection channel, but not for HPV18. Given its modest standalone discriminatory performance and the selected nature of the histologically verified cohort, ΔCt should be considered an exploratory marker rather than a validated triage tool. Prospective validation is required to determine whether it provides incremental clinical value.